Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have now examined the structure of the inactive complex in more detail using single particle analysis of negatively stained myosin V molecules. Averaged images show considerable detail. The motor domain and lever arm of each molecule bends acutely back upon its stalk and each of the motor domains contact a lobe of the globular tail domain (GTD). The point of contact at the motor domain is a surface loop located near the nucleotide binding site that contains four negatively charged amino acids that are conserved in the myosin V family, but not necessarily in other myosin superfamily members. A truncated HMM-like fragment of myosin V lacking the distal coiled-coil domain of the stalk and the GTD has a high ATPase activity that is not affected by calcium concentration. Addition of a GST-GTD dimeric fusion protein inhibits the ATPase activity of myosin V HMM in the absence, but not the presence of calcium. Examination of the HMM-GST-GTD complex in the absence of calcium shows that the two motor domains of HMM have folded back and contacted the two GTD domains to form a structure remarkably like that seen in the intact molecule. A mutant GTD in which several charged groups have been neutralized does not inhibit the activity of HMM. [unreadable] [unreadable] We have expressed and characterized the kinetic and motile behavior of fragments of human myosin Vc. The detailed kinetic characterization suggests that this motor has a low duty ratio unlike that of the better characterized myosin Va isoforms and is not likely to be processive. Attempts to see processive behavior using single molecule TIRF assays have been unsucessful.[unreadable] [unreadable] We collaborated with Dr. Peter Takizawa's group to examine the in vitro movement of two yeast myosin V isoforms and observe its molecular structure by rotary shadowing electron microscopy. Our data showed that myo4p is monomeric whereas myo2p is dimeric. Furthermore myo2p is weakly processive.[unreadable] [unreadable] We have used a fluorescently-labeled ATP analog termed deac-aminoATP to simulataneously visualize the stepping of fluorescently-labeled myosin V HMM and binding and dissociation of the nucleotide using the FIONA method. These studies show that there is a direct one to one coupling of myosin movement and nucleotide binding/dissociation and that there is strong gating between the kinetics of the two heads such that ADP release from the lead head is essentially prevented as long as the trail head is still attached.